Compound, optical resolution method, and derivative of an optical isomer of an amino acid

ABSTRACT

Disclosed is a compound represented by chemical formula (1): 
                         
or chemical formula (2):

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional application and claims priorityunder 35 U.S.C. 120 to U.S. patent application Ser. No. 14/472,920 filedon Aug. 29, 2014, which claims priority under 35 U.S.C. 119 to JapanesePatent Application No. 2013-181305 filed on Sep. 2, 2013. The entirecontents of the foregoing applications are incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

One aspect of the present invention relates to at least one of acompound, an optical resolution method, and a derivative of an opticalisomer of an amino acid.

2. Description of the Related Art

Conventionally, it has been considered that amino acids present in aliving body of a higher animal are only L-amino acids. However, it isfound that D-amino acids are also present in a living body, due todevelopment of an analysis technique in recent years.

From the viewpoint of elucidating of a physiological role of an aminoacid in a living body, development and study of an analytical techniquefor precisely quantifying an amino acid (see, for example, K. Shimbo etal., Anal. Chem., 2009, 81, 5172-5179; K. Shimbo et al., Rapid Commun.Mass Spectrom., 2009, 23, 1483-1492; and S. A. Cohen et al., Anal.Biochem., 1993, 211, 279-287) or a D-amino acid and an L-amino acid(see, for example, R. J. Reischl et al., J. Chromatogra. A., 2012, 1269,262-269) are being advanced by utilizing high-performance liquidchromatography (HPLC).

However, an optical resolution method as described in K. Shimbo et al.,Anal. Chem., 2009, 81, 5172-5179; K. Shimbo et al., Rapid Commun. MassSpectrom., 2009, 23, 1483-1492; S. A. Cohen et al., Anal. Biochem.,1993, 211, 279-287; or R. J. Reischl et al., J. Chromatogra. A., 2012,1269, 262-269 has a problem that a performance and a sensitivity ofoptical resolution are low. Furthermore, in order to prepare ananalytical sample, it is necessary to conduct a reaction between amixture of optical isomers of an amino acid and a compound for opticalresolution for a comparatively long period of time while heat is appliedthereto.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided acompound represented by chemical formula (1):

or chemical formula (2):

According to another aspect of the present invention, there is providedan optical resolution method, including a first step of mixing opticalisomers of an amino acid with the compound as described above to obtainderivatives of the optical isomers.

According to another aspect of the present invention, there is provideda derivative of an optical isomer of an amino acid obtained by theoptical resolution method as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is one example of a synthetic scheme for a compound for opticalresolution according to the present embodiment.

FIG. 2 is another example of a synthetic scheme for a compound foroptical resolution according to the present embodiment.

FIG. 3 is a flow diagram of one example of a optically resolving methodthat uses a compound for optical resolution according to the presentembodiment.

FIG. 4 is one example of a result of HPLC-FD analysis that uses acompound for optical resolution according to the present embodiment.

FIG. 5 is one example of a result of HPLC-MS analysis that uses acompound for optical resolution according to the present embodiment.

FIG. 6 is a schematic diagram for illustrating one example of an effectof a compound for optical resolution according to the presentembodiment.

FIG. 7 is a schematic diagram for illustrating another example of aneffect of a compound for optical resolution according to the presentembodiment.

FIG. 8 is a schematic diagram for illustrating another example of aneffect of a compound for optical resolution according to the presentembodiment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

An embodiment of the present invention will be described in detailbelow. Here, although an embodiment that uses an amino acid as a targetfor optical resolution is primarily described in the presentspecification, it is possible for a compound for optical resolutionaccording to an embodiment of the present invention to conduct opticalresolution at a high sensitivity, even for another target for opticalresolution such as an amine, an alcohol, a thiol, or a carboxylic acid.

A Compound for Optical Resolution

The inventors have found that a performance of optical resolution of anamino acid is significantly improved by using a reagent for opticalresolution (or a kit for optical resolution) that includes a compoundrepresented by structural formula (1) or structural formula (2)described below to derivatize a mixture of optical isomers of an aminoacid in a technique of optical resolution of an amino acid or the likein an HPLC method.

A detail of a reason why a performance of optical resolution in an HPLCmethod is improved by using a compound with a structural formula (1) orstructural formula (2) to derivative an amino acid is underinvestigation. However, as derivatization is conducted by using thesecompounds, it is possible to conduct optical resolution of an obtainedamino acid derivative in an HPLC method at a high sensitivity.Furthermore, it has also been found that it is possible for a compoundwith structural formula (1) or structural formula (2) described above toderivative an amino acid in a short period of time and an obtained aminoacid derivative has excellent heat stability and light stability.

An Example of Synthesis of a Compound for Optical Resolution withStructural Formula (1)

FIG. 1 illustrates one example of a synthetic scheme of a compound foroptical resolution according to the present embodiment. A specificexample of synthesis of a compound for optical resolution withstructural formula (1) will be described below, with reference to FIG.1.

4.32 parts of diethyl ethoxymethylenemalonate were added to 2.45 partsof p-anisidine, and heating and stirring thereof were conducted at 110°C. for 2 hours by using an oil bath under a nitrogen stream. Then, 20 mlof diphenyl ether were added thereto, and reflux thereof was conductedfor 1 hour by using a mantle heater. After standing to cool to roomtemperature, hexane was added to a reaction solution to filtrate andobtain a precipitate and an obtained product was washed with acetone.

15 ml of a 1 M aqueous solution of sodium hydroxide were added to thisproduct and reflux thereof was conducted for 1 hour. After a reactionsolution was acidized with an aqueous solution of hydrochloric acid, aprecipitate was filtrated and obtained and an obtained product waswashed with water.

Then, 20 ml of diphenyl ether were added to this product, and refluxthereof was conducted for 1.5 hours under a nitrogen stream by using amantle heater. After standing to cool to room temperature, hexane wasadded to a reaction solution to filtrate and obtain a precipitate and anobtained product was washed with acetone to obtain a white powder(S100). An obtained powder was identified by ¹H NMR as6-methoxyquinolin-4(1H)-one. Here, in the present specification. ¹H NMRwas conducted at a strength of electric field of 400 mega-hertz (MHz).

Here, a result of ¹H NMR measurement was: ¹H NMR (dmso): δ 3.817 (3H,s), 5.975 (1H, d, J=7.6 Hz), 7.270 (1H, dd, J=3.2, 9.2 Hz), 7.477 (1H,s), 7.492 (1H, d, J=5.6 Hz), 7.818 (1H, s), 11.676 (1H, s).

526 parts of obtained 6-methoxyquinolin-4(1H)-one, 107 parts ofpotassium hydroxide, and 798 parts of ethylene carbonate were dissolvedin 5 ml of N,N-dimethylformamide (DMF), and heating and stirring thereofwere conducted at 130° C. overnight by using an oil bath under anitrogen stream. After completion of a reaction, a solvent in a reactionsolution was removed under a reduced pressure. An obtained residue wasfractionated by silica gel column chromatography (developing solvent;chloroform:methanol=20:1→10:1) to obtain a white powder (S110). Anobtained powder was identified by ¹H NMR as1-(2-hydroxyethyl)-6-methoxyquinolin-4(1H)-one. Here, if is preferablefor a white powder of 1-(2-hydroxyethyl)-6-methoxyquinolin-4(1H)-one tobe subjected to a recrystallization process that uses chloroform orethanol, in order to improve a purity thereof, and to be supplied to anext step as a needle crystal.

Here, a result of ¹H NMR measurement was: ¹H NMR (dmso): δ 3.695 (2H, q,J=5.6, 5.2 Hz), 3.835 (3H, s), 4.270 (2H, t, J=5.2 Hz), 4.926 (1H, t,J=5.2 Hz), 5.981 (1H, d, J=7.6 Hz), 7.318 (1H, dd, J=2.8, 9.4 Hz), 7.599(1H, d, J=3.6 Hz), 7.708 (1H, d, J=9.6 Hz), 7.824 (1H, d, J=7.6 Hz).

1 part by mass of di(N-succinimidyl) carbonate was dissolved in 10 ml ofdichloromethane and 2 ml of pyridine was added thereto. After1-(2-hydroxyethyl)-6-methoxyquinolin-4(1H)-one that had preliminarilybeen dissolved in dichloromethane was dropped into this solution for 1hour, stirring thereof was subsequently conducted overnight. Aftercompletion of a reaction, a reaction solution was washed with 0.1 M ofhydrochloric acid and a saturated saline and an organic phase was driedover magnesium sulfate. After magnesium sulfate was filtrated andseparated, a solvent was removed under a reduced pressure. An obtainedresidue was fractionated by silica gel chromatography (developingsolvent; chloroform:methanol=30:1) to obtain a white powder (S120). Anobtained powder was identified by ¹H NMR as2,5-dioxopyrolidin-1-yl(2-(6-methoxy-4-oxoquinolin-1(4H)-yl)ethyl)carbonate.

Here, a result of ¹H NMR measurement was: ¹H NMR (CD₃CN): δ 2.716 (4H,s), 3.882 (3H, s), 4.499 (2H, t, J=5, 5.2 Hz), 4.644 (2H, t, J=4.8, 5Hz), 6.060 (1H, d, J=7.6 Hz), 7.324 (1H, dd, J=3.2, 9.2 Hz), 7.562 (1H,d, J=9.6 Hz), 7.655 (1H, d, J=7.6 Hz), 7.707 (1H, d, J=2.8 Hz).

An Example of Synthesis of a Compound for Optical Resolution withStructural Formula (2)

FIG. 2 illustrates another example of a synthetic scheme of a compoundfor optical resolution according to the present embodiment. A specificexample of synthesis of a compound for optical resolution withstructural formula (2) described above will be described below, withreference to FIG. 2.

500.5 parts by mass of 6-methoxyquinolin-4(1H)-one, 611.1 parts by massof potassium carbonate, and 500 μL of ethyl bromoacetate were dissolvedin 5 ml of DMF and stirring thereof was conducted overnight. Aftercompletion of a reaction, chloroform was added to a reaction solution toconduct filtration and separation, and an obtained product was driedover magnesium sulfate.

After magnesium sulfate was filtrated and separated, an organic solventwas removed under a reduced pressure. An obtained residue wasfractionated by silica gel chromatography (developing solvent;chloroform:methanol=15:1→10:1) to obtain a white powder (S200). Anobtained powder was identified by ¹H NMR as ethyl2-(6-methoxy-4-oxoquinolin-1(4H)-yl)acetate.

Here, a result of ¹H NMR measurement was: ¹H NMR (dmso); δ 1.196 (3H, t,J=7.6 Hz), 3.836 (3H, s), 4.159 (2H, dd, J=7.2, 14 Hz), 5.178 (2H, s),6.039 (1H, d, J=7.6 Hz), 7.318 (1H, dd, J=2.8, 9.2 Hz), 7.433 (1H, d,J=9.2 Hz), 7.587 (1H, d, J=3.2 Hz), 7.871 (1H, d, J=7.6 Hz).

650 parts by mass of obtained ethyl2-(6-methoxy-4-oxoquinolin-1(4H)-yl)acetate were dissolved in 5 ml of a1 M aqueous solution of sodium hydroxide and stirring thereof wasconducted overnight. After completion of a reaction, 3 M of hydrochloricacid were added to a reaction solution to filtrate and obtain aprecipitate, and subsequently washing with water was conducted to obtaina white powder (S210). An obtained powder was identified by ¹H NMR as2-(6-methoxy-4-oxoquinolin-1(4H)-yl)acetic acid.

Here, a result of ¹H NMR measurement was: ¹H NMR (dmso): δ 3.835 (3H,s), 5.062 (2H, s), 6.024 (1H, d, J=7.6 Hz), 7.324 (1H, dd, J=3.2, 9.4Hz), 7.439 (1H, d, J=9.2 Hz), 7.587 (1H, d, J=2.8 Hz), 7.873 (1H, d,J=7.6 Hz), 13.304 (1H, s).

101 parts by mass of obtained 2-(6-methoxy-4-oxoquinolin-1(4H)-yl)aceticacid were dissolved in 700 μL of thionyl chloride and stirring thereofwas conducted at 0° C. for 2 hours. After completion of a reaction,thionyl chloride was removed under a reduced pressure and subsequentlyhexane was added to filtrate and obtain a precipitate, so that a yellowpowder was obtained (S220). An obtained powder was identified by ¹H NMRas 2-(6-methoxy-4-oxoquinolin-1(4H)-yl)acetic acid chloride.

Here, a result of ¹H NMR measurement was: ¹H NMR (dmso): δ 3.893 (3H,s), 5.338 (2H, s), 6.611 (1H, d, J=7.2 Hz), 7.520 (1H, dd, J=2.8, 9.4Hz), 7.631 (1H, d, J=3.2 Hz), 7.720 (1H, d, J=9.6 Hz), 8.310 (1H, d,J=7.2 Hz).

An Optically Resolving Method

Next, a method for optically resolving a mixture of optical isomers ofan amino acid by using a compound for optical resolution according tothe present embodiment will be described.

FIG. 3 illustrates a flow diagram of one example of an opticallyresolving method that uses a compound for optical resolution accordingto the present embodiment.

An optically resolving method according to the present embodimentincludes a first step (S300) wherein a reagent for optical resolutionand a mixture of optical isomers are mixed to obtain a derivative of themixture of optical isomers, and a second step (S310) wherein thederivative is separated into respective optical isomers by using columnchromatography.

At a first step of S300, a reagent for optical resolution and a mixtureof optical isomers are mixed to obtain a derivative of the mixture ofoptical isomers.

A mixing method is not particularly limited, and for example, if ispossible to conduct mixing and reacting thereof at a room temperaturefor about 1 minute to provide an amino acid derivative. In a case wherea conventional reagent for optical resolution is used, it has beennecessary to apply heat to and react for a long period of time an aminoacid and such a compound for optical resolution in order to prepare ananalytical sample. However, in a case where a compound for opticalresolution according to the preset embodiment is used, mixing thereof isconducted at a room temperature for about 1 minute so that it ispossible to obtain an amino acid derivative, and it is considered thatsuch a method is convenient.

Then, at a second step of S310, a mixture of optical isomers as obtainedat the first step is separated into respective optical isomers by usingcolumn chromatography.

For a column for column chromatography, it is possible to use aconventional and commercially available column.

Next, an embodiment of the present invention will be described in moredetail by providing specific embodiments.

A First Embodiment

In a first embodiment, an embodiment will be described for confirmingthat an amino acid derivative for which a compound for opticalresolution with structural formula (1) described above is used iscapable of optical resolution at a good optical resolution performanceby HPLC analysis that uses a conventional column.

A compound for optical resolution according to the present embodiment isadded to a solution that contains an amino acid as a measurement targetand a reaction is caused at a room temperature for about 1 minute, sothat it is possible to provide an amino acid derivative for an HPLCmeasurement.

Specifically, 10 μL of a 40 mM boric acid buffer solution (pH=8) and 5μL of a solution of a compound for optical resolution with structuralformula (1) described above (concentration: 40 mM) are added to 10 μL ofan amino acid solution with a predetermined concentration, for example,a concentration of 50 μM, 100 μM, 125 μM, or the like, and mixingthereof is conducted by a vortex mixer for 1 minute, so that it ispossible to derivative an amino acid.

In the first embodiment, 10 μL of a 40 mM boric acid buffer solution(pH=8) and 5 μL of a solution of a compound for optical resolution withformula (1) described above (concentration: 40 mM) were added to 10 μLof an amino acid solution with a concentration of 100 μM, 125 μM, or thelike, and mixing thereof was conducted by a vortex mixer for 1 minute,so that an amino acid was derivatized. 475 μL of a 0.55 aqueous solutionof trifluoroacetic acid were added to an obtained solution thatcontained an amino acid derivative to stop a reaction and prepare asample for HPLC analysis. HPLC analysis was applied to 0.1 μL of anobtained sample. For fluorescence analysis, an excitation wavelength was243 nm and a fluorescence wavelength was 380 nm, while, for massspectrometry, for example, mass spectrometry for alanine was conductedfor a product ion with a mass-to-charge ratio (M/Z) of 114 produced froma precursor ion with an M/Z of 335.

Analysis conditions for HPLC analysis were:

a column: QN-1AX (1.5 mm i.d.×150 mm);

a flow rate: 200 μL/min;

a temperature: 25° C.;

mobile phase 1 (MP1): 0.02% formic acid methanol/acetonitrile=50/50;

mobile phase 2 (MP2): 0.05% formic acid methanol/acetonitrile=50/50;

mobile phase 3 (MP3): 0.2% formic acid methanol/acetonitrile=50/50;

mobile phase 4 (MP4): 10 mM ammonium formatemethanol/acetonitrile=50/50; and

mobile phase 5 (MP5): 0.5% formic acid methanol/acetonitrile=50/50.

Table 1 illustrates a summary of kinds of each amino acid and a mobilephase, an elution time for HPLC analysis, elation orders of an L-aminoacid and a D-amino acid, and a separation factor α.

TABLE 1 First Second Early Kind of Kind of elution elution eluted aminomobile time R_(t1) time R_(t2) optical Separation acid phase (min) (min)isomer factor α Ala MP1 9.15 10.477 D-body 1.157 Asn MP1 13.48 14.733D-body 1.098 Gln MP1 12.28 13.73 D-body 1.125 Trp MP2 9.52 11.933 D-body1.274 Asp MP5 13.097 13.937 D-body 1.068 Glu MP3 5.53 6.58 D-body 1.22Ser MP2 11.733 13.837 D-body 1.191 Thr MP2 9.12 11.51 D-body 1.284allo-Thr MP2 12.033 16.253 D-body 1.372 Tyr MP2 10.403 12.137 D-body1.179 Phe MP1 9.25 10.77 D-body 1.179 Val MP1 7.943 10.213 D-body 1.313Leu MP1 6.597 8.208 D-body 1.285 Ile MP1 7.44 9.633 D-body 1.325 all-IleMP1 7.813 10.17 D-body 1.33 Cys MP2 20.81 24.97 D-body 1.207 Met MP112.593 15.357 D-body 1.232 Cystine MP5 13.643 19.173 D-body 1.427(CysCys) Lys MP1 6.723 8.073 D-body 1.224 His MP4 5.323 6.127 D-body1.174

As is clear from Table 1, a separation factor α of an amino acidderivatized by a compound for optical resolution according to thepresent embodiment was greater than or equal to 1.07 independently of akind thereof (for example, a neutral amino acid, an acidic amino acid, ahydrophobic amino acid, a sulfur-containing amino acid, a basic aminoacid, or the like).

Furthermore, a defection limit of an optically resolving method thatused a compound for optical resolution according to the presentembodiment was at a femt mol order—an atto mol order per an amount ofinjection and was very highly sensitive as compared to that of aconventional method.

From the above, it was found that a compound for optical resolutionaccording to the present embodiment was used to prepare an amino acidderivative and this was analyzed by HPLC, so that it was possible tooptically resolve a mixture of optical isomers of an amino acid at ahigh resolution and a high detection sensitivity by a convenient method.

A Second Embodiment

Furthermore, an embodiment will be described for evidencing that aneffect of a compound for optical resolution according to the presentembodiment is independent on a kind of a column and is also capable ofbeing applied to another conventional column.

Predetermined amino acids were optically resolved by a method similar tothat of the first embodiment except that analysis conditions for HPLCanalysis were changed to the following and kinds of amino acids werechanged partially.

Analysis conditions for HPLC analysis were:

a column: QD-1AX (1.5 mm i.d.×150 mm);

a flow rate: 200 μL/min;

a temperature: 25° C.;

mobile phase 1 (MP1): 0.02% formic acid methanol/acetonitrile=50/50;

mobile phase 2 (MP2): 0.05% formic acid methanol/acetonitrile=50/50;

mobile phase 3 (MP3): 0.25% formic acid methanol/acetonitrile=50/50;

mobile phase 4 (MP4): 10 mM ammonium formate that contained 0.32% formicacid methanol/acetonitrile=20/80;

mobile phase 5 (MP5): 0.5% formic acid methanol/acetonitrile=50/50; and

mobile phase 6 (MP6): 1% formic acid methanol/acetonitrile=50/50.

Table 2 illustrates a summary of kinds of each amino acid and a mobilephase, an elation time for HPLC analysis, elation orders of an L-aminoacid and a D-amino acid, and a separation factor α.

TABLE 2 First Second Early Kind of Kind of elution elution eluted aminomobile time R_(t1) time R_(t2) optical Separation acid phase (min) (min)isomer factor α Ala MP1 9.307 11.757 L-body 1.285 Asn MP1 14.037 15.82L-body 1.134 Gln MP1 12.99 15.557 L-body 1.209 Trp MP2 10.553 13.677L-body 1.317 Asp MP3 14.483 16.567 L-body 1.151 Glu MP3 6.577 8.737L-body 1.368 Ser MP2 12.413 16.623 L-body 1.359 Thr MP2 10.317 13.873L-body 1.37 allo-Thr MP2 12.833 19.237 L-body 1.528 Tyr MP2 10.913 14.18L-body 1.32 Phe MP1 15.657 20.197 L-body 1.304 Val MP1 7.74 10.43 L-body1.382 Leu MP1 6.423 8.743 L-body 1.405 Ile MP1 7.163 9.983 L-body 1.436all-Ile MP1 7.427 10.193 L-body 1.411 Cys MP5 7.21 8.94 L-body 1.266 MetMP1 13.177 17.66 L-body 1.359 Cystine MP6 4.6 6.833 L-body 1.573(CysCys) Arg MP4 6.087 7.037 L-body 1.18 Lys MP1 8.06 9.48 L-body 1.193His MP4 10.14 12.67 L-body 1.267

Furthermore, predetermined amino acids were optically resolved by amethod similar to that of the first embodiment except that analysisconditions for HPLC analysis were changed to the following and kinds ofamino acids were changed partially.

Analysis conditions for HPLC analysis were:

a column: Sumichiral OA-3200S (1.5 mm i.d.×250 mm);

a flow rate: 200 μL/min;

a temperature: 25° C.;

mobile phase 1 (MP1): 0.05% formic acid methanol/acetonitrile=50/50;

mobile phase 2 (MP2): 0.1% formic acid methanol/acetonitrile=50/50;

mobile phase 3 (MP3): 0.05% formic acid methanol/acetonitrile=50/50; and

mobile phase 4 (MP4): 0.25% formic acid methanol/acetonitrile=50/50.

Table 3 illustrates a summary of kinds of each amino acid and a mobilephase, an elution time for HPLC analysis, elution orders of an L-aminoacid and a D-amino acid, and a separation factor α.

TABLE 3 First Second Early Kind of Kind of elution elution eluted aminomobile time R_(t1) time R_(t2) optical Separation acid phase (min) (min)isomer factor α Ala MP1 11.097 11.473 D-body 1.038 Asn MP2 12.4949713.033 D-body 1.048 Ser MP2 12.23 12.95 D-body 1.065 Thr MP2 9.333 10.04D-body 1.087 Val MP1 8.44 8.89 D-body 1.062 Leu MP1 7.26 8.003 D-body1.123 Ile MP1 7.803 8.227 D-body 1.064 all-Ile MP1 7.783 8.63 1.129 CysMP2 25.877 26.8 D-body 1.037 Met MP1 11.953 12.93 D-body 1.091 CystineMP3 19.573 20.157 D-body 1.032 (CysCys) Lys MP1 11.58 12.413 D-body 1.08

Moreover, predetermined amino acids were optically resolved by a methodsimilar to that of the first embodiment except that analysis conditionsfor HPLC analysis were changed to the following and kinds of amino acidswere changed partially.

Analysis conditions for HPLC analysis were:

a column: Sumichiral OA-4700SR (1.5 mm i.d.×250 mm);

a flow rate: 200 μL/min;

a temperature: 25° C.;

mobile phase 1 (MP1): 0.02% formic acid methanol/acetonitrile=50/50; and

mobile phase 2 (MP2): 0.05% formic acid methanol/acetonitrile=50/50.

Table 4 illustrates a summary of kinds of each amino acid and a mobilephase, an elution time for HPLC analysis, elation orders of an L-aminoacid and a D-amino acid, and a separation factor α.

TABLE 4 First Second Early Kind of Kind of elution elution eluted aminomobile time R_(t1) time R_(t2) optical Separation acid phase (min) (min)isomer factor α allo-Thr MP2 11.763 12.507 D-body 1.071 Phe MP1 13.46314.433 D-body 1.079 Val MP1 8.713 9.383 D-body 1.089 Leu MP1 6.747 7.62D-body 1.157 Ile MP1 7.54 8.357 D-body 1.129 all-Ile MP1 7.875 8.81D-body 1.143 Met MP1 12.183 13.363 D-body 1.107 Lys MP1 8.257 9.277D-body 1.145

Moreover, predetermined amino acids were optically resolved by a methodsimilar to that of the first embodiment except that analysis conditionsfor HPLC analysis were changed to the following and kinds of amino acidswere changed partially.

Analysis conditions for HPLC analysis were:

a column: KSAACSP-00S (1.5 mm i.d.×250 mm);

a flow rate: 200 μL/min;

a temperature: 25° C.;

mobile phase 1 (MP1): 0.05% formic acid methanol/acetonitrile=50/50;

mobile phase 2 (MP2): 0.1% formic acid methanol/acetonitrile=50/50; and

mobile phase 3 (MP3): 5 mM solution of ammonium formate in methanol(flow rate: 150 μL/min).

Table 5 illustrates a summary of kinds of each amino acid and a mobilephase, an elution time for HPLC analysis, elution orders of an L-aminoacid and a D-amino acid, and a separation factor α.

TABLE 5 First Second Early Kind of Kind of elution elution eluted aminomobile time R_(t1) time R_(t2) optical Separation acid phase (min) (min)isomer factor α Pro MP1 13.82 14.873 L-body 1.084 Trp MP2 7.577 8.24L-body 1.104 Ser MP2 13.043 13.583 D-body 1.046 Thr MP2 9.473 9.977D-body 1.061 allo-Thr MP2 12.373 13.003 L-body 1.056 Phe MP1 12.35713.133 L-body 1.07 Val MP1 8.287 8.887 L-body 1.086 Ile MP1 7.89 8.597L-body 1.107 all-Ile MP1 7.5 8.133 L-body 1.102 Met MP1 11.303 11.737L-body 1.091 Arg MP3 11.607 11.96 L-body 1.04 Lys MP1 12.92 13.877L-body 1.082

As is clear from results of Table 1 and Table 2 to Table 5, a separationfactor α of an amino acid derivatized by a compound for opticalresolution according to the present embodiment was greater than or equalto 1 independently of a kind of a column or a kind of an amino acid.

From the above, it was found that a compound for optical resolutionaccording to the present embodiment was used to prepare an amino acidderivative and this was analyzed by HPLC that used a conventionalcolumn, so that it was possible to optically resolve a mixture ofoptical isomers of an amino acid at a high resolution and a highdetection sensitivity by a convenient method.

A Third Embodiment

In a third embodiment, an embodiment will foe described for confirmingthat an amino acid derivative for which a compound for opticalresolution with structural formula (2) described above is used iscapable of optical resolution by HPLC analysis that uses a conventionalcolumn.

Alanine was derivatized by a method similar to that of the firstembodiment except that a compound with structural formula (2) was usedas a compound for optical resolution.

HPLC analysis was applied to 0.1 μL of an obtained sample. Forfluorescence analysis, an excitation wavelength was 243 nm and afluorescence wavelength was 380 nm, while, for mass spectrometry, massspectrometry was conducted for a product ion with a mass-to-charge ratio(M/Z) of 259 produced from a precursor ion with an M/Z of 305.

Analysis conditions for HPLC analysis were:

a column: QN-AX (1.5 mm i.d.×150 mm);

a flow rate: 200 μL/min;

a temperature: 25° C.; and

a mobile phase: 0.05% formic acid methanol/acetonitrile=50/50.

FIG. 4 illustrates one example of a result of HPLC-FL analysis and FIG.5 illustrates one example of a result of HPLC-MS analysis. In FIG. 4, atransverse axis indicates an elution time and a longitudinal axisindicates a fluorescence intensity. Furthermore, in FIG. 5, a transverseaxis indicates an elution time and a longitudinal axis indicates an ionintensity. Furthermore, FIG. 4 and FIG. 5 indicate, in combination,results of cases where only an L-body or a D-body was subjected to asimilar analysis.

As is clear from FIG. 4 and FIG. 5, it was found in any result ofanalysis that a compound for optical resolution according to the presentembodiment was used so that it was possible to separate a mixture ofoptical isomers completely.

Furthermore, a detection limit of an optically resolving method thatused a compound for optical resolution according to the presentembodiment was at a femt mol order—an atto mol order per an amount ofinjection and was very highly sensitive as compared to that of aconventional method.

From the above, if was found that a compound for optical resolutionaccording to the present embodiment was used to prepare an amino acidderivative and this was analyzed by HPLC, so that it was possible tooptically resolve a mixture of optical isomers of an amino acid at ahigh resolution and a high detection sensitivity by a convenient method.

A Fourth Embodiment

It is very important to derivatize an amino acid easily for a shortperiod of time in a technical field where an amino acid is derivatizedand analyzed by HPLC. In a fourth embodiment, an embodiment will bedescribed for confirming that it is possible for a compound for opticalresolution according to the present embodiment to derivatize an aminoacid for a short period of time.

10 μL of a 40 mM boric acid buffer solution (pH=8) and 5 μL of asolution of a compound for optical resolution with structural formula(1) described above (concentration: 40 mM) were added to 10 μL of anL-alanine solution with a concentration of 50 μM. This solution wasmixed by a vortex mixer for 1, 2, 5, or 10 minutes to derivative anamino acid. 475 μL of a 0.5% aqueous solution of trifluoroacetic acidwere added to an obtained solution that contained an amino acidderivative to stop a reaction and prepare a sample for HPLC analysis.Furthermore, for a comparative embodiment, 475 μL of a 0.5% aqueoussolution of trifluoroacetic acid were added without passing throughmixing by a vortex mixer to prepare a sample for HPLC analysis.

HPLC analysis was applied to 0.1 μL of an obtained sample similarly tothe first embodiment. For fluorescence analysis, an excitationwavelength was 243 nm and a fluorescence wavelength was 380 nm, while,for mass spectrometry, mass spectrometry was conducted for a product ionwith a mass-to-charge ratio (M/Z) of 114 produced from a precursor ionwith an M/Z of 335.

A schematic diagram for illustrating one example of an effect of acompound for optical resolution according to the present embodiment isillustrated in FIG. 6. In FIG. 6, a transverse axis is for a mixing timefor a compound for optical resolution and L-alanine and a longitudinalaxis is for a peak height (μV) originating from an L-alanine derivative.

As illustrated in FIG. 6, a change of a peak height was hardly observedfor a sample with a mixing time of 1 minute to 10 minutes whereas achange of a peak height was observed between a sample with a mixing timeof 0 minutes and a sample with that of 1 minute to 10 minutes.Accordingly, it was found that it was possible for a compound foroptical resolution according to one present embodiment to derivatize anamino acid by only being mixed with such an amino acid for about 1minute.

A Fifth Embodiment

In a fifth embodiment, an embodiment will be described for confirmingthat an amino acid derivative obtained by reacting a compound foroptical resolution according to the present embodiment with an aminoacid has an excellent heat stability.

10 μL of a 40 mM boric acid buffer solution (pH=8) and 5 μL of asolution of a compound for optical resolution with structural formula(1) described above (concentration: 40 mM) were added to 10 μL of anL-alanine solution with a concentration of 50 μM. This solution wasmixed by a vortex mixer for 1 minute to derivative an amino acid. 475 μLof a 0.5% aqueous solution of trifluoroacetic acid were added to anobtained solution that contained an amino acid derivative to prepare asample for HPLC analysis.

50 μL of an obtained sample for HPLC analysis were heated for 5, 10, 20,30, or 60 minutes by using an aluminum heater that was set at 50° C.preliminarily. HPLC analysis was applied to 0.1 μL of a sample afterheating similarly to the first embodiment. For fluorescence analysis, anexcitation wavelength was 243 nm and a fluorescence wavelength was 380nm, while, for mass spectrometry, mass spectrometry was conducted for aproduct ion with a mass-to-charge ratio (M/Z) of 114 produced from aprecursor ion with an M/Z of 335.

A schematic diagram for illustrating another example of an effect of acompound for optical resolution according to the present embodiment isillustrated in FIG. 7. In FIG. 7, a transverse axis is for a heatingtime for an L-alanine derivative and a longitudinal axis is tor a peakheight (μV) originating from such an L-alanine derivative.

As illustrated in FIG. 7, reduction of an L-alanine derivative was notconfirmed within a range of a heating time in the present embodiment.Accordingly, it was found that an amino acid derivative obtained byusing a compound for optical resolution according to the presentembodiment has an excellent heat stability.

A Sixth Embodiment

In a sixth embodiment, an embodiment will be described for confirmingthat an amino acid derivative obtained by reacting a compound foroptical resolution according to the present embodiment with an aminoacid has an excellent light stability.

10 μL of a 40 mM boric acid buffer solution (pH=8) and 5 μL of asolution of a compound for optical resolution with structural formula(1) described above (concentration: 40 mM) were added to 10 μL of anL-alanine solution with a concentration of 50 μM. This solution wasmixed by a vortex mixer for 1 minute to derivatize an amino acid. 475 μLof a 0.5% aqueous solution of trifluoroacetic acid were added to anobtained solution that contained an amino acid derivative to prepare asample for HPLC analysis.

An obtained sample for HPLC analysis was stood for 0.5, 1, 2, 3, or 5hours under light irradiation by a fluorescent lamp. HPLC analysis wasapplied to 0.1 μL of a sample after standing similarly to the firstembodiment. For fluorescence analysis, an excitation wavelength was 243nm and a fluorescence wavelength was 380 nm, while, for massspectrometry, mass spectrometry was conducted for a product ion with amass-to-charge ratio (M/Z) of 114 produced from a precursor ion with anM/Z of 335.

A schematic diagram for illustrating another example of an effect of acompound for optical resolution according to the present embodiment isillustrated in FIG. 8. In FIG. 8, a transverse axis is for anirradiation time for an L-alanine derivative and a longitudinal axis isfor a peak height (μV) originating from such an L-alanine derivative.

As illustrated in FIG. 8, reduction of an L-alanine derivative was notconfirmed within ranges of an irradiation intensity and an irradiationtime in the present embodiment. Accordingly, it was found that an aminoacid derivative obtained by using a compound for optical resolutionaccording to the present embodiment has an excellent light stability.

From the above, it was found that it was possible to mix a compound foroptical resolution according to the present embodiment with a mixture ofoptical isomers of an amino acid to prepare an amino acid derivative,and this was analyzed by HPLC in such a manner that it was possible tooptically resolve a mixture of optical isomers at a high resolution anda high detection sensitivity by a convenient method. At this time, itwas found that a mixture of optical isomers of an amino acid wasderivatized by mixing at a room temperature for about 1 minute.Furthermore, it was found that an obtained amino acid derivative wasexcellent in a heat stability and a light stability.

Appendix

<An Illustrative Embodiment(s) of a Compound for Optical Resolution, aReagent for Optical Resolution, an Optically Resolving Method, and anOptical Isomer>

At least one illustrative embodiment of the present invention may relateto a compound for optical resolution, a reagent for optical resolution,an optically resolving method, and an optical isomer.

At least one illustrative embodiment of the present invention may aim atproviding a compound for optical resolution that is capable of opticallyresolving a mixture of optical isomers of an amino acid conveniently ata high sensitivity, against a problem as described above.

At least one illustrative embodiment of the present invention may beprovided as a compound for optical resolution that is a structuralformula (1):

or structural formula (2):

Illustrative embodiment (1) is a compound tor optical resolution that isstructural formula (1):

or structural formula (2):

Illustrative embodiment (2) is a reagent for optical resolution, thatincludes the compound for optical resolution as described inillustrative embodiment (1).

Illustrative embodiment (3) is a method for optically resolving amixture of optical isomers of an amino acid, that uses the reagent foroptical resolution as described in illustrative embodiment (2).

Illustrative embodiment (4) is the method for optically resolving asdescribed in illustrative embodiment (3), that includes a first step ofmixing the reagent for optical resolution and the mixture of opticalisomers to obtain a derivative of the mixture of optical isomers.

Illustrative embodiment (5) is the method for optically resolving asdescribed in illustrative embodiment (4), wherein the first stepincludes a step of mixing at a room temperature for one minute.

Illustrative embodiment (6) is the method for optically resolving asdescribed in illustrative embodiment (4) or (5), that includes a secondstep of separating the derivative into respective optical isomers byusing column chromatography after the first step.

Illustrative embodiment (7) is an optical isomer of an amino acid thatis obtained by the method for optically resolving as described in anyone of illustrative embodiments (4) to (6).

According to at least one illustrative embodiment of the presentinvention, it may be possible to provide a compound for opticalresolution that is capable of optically resolving a mixture of opticalisomers of an amino acid conveniently at a high sensitivity.

Although the illustrative embodiment(s) and specific example(s) of thepresent invention have been described with reference to the accompanyingdrawings, the present invention is not limited to any of theillustrative embodiment(s) and specific example(s) and the illustrativeembodiment(s) and specific example(s) may be altered, modified, orcombined without departing from the scope of the present invention.

What is claimed is:
 1. An optical resolution method, comprising:reacting a mixture of optically active amino acids with a compoundrepresented by chemical formula (1):

or chemical formula (2):

to obtain a derivatized amino acid mixture; and subjecting thederivatized amino acid mixture to column chromatography to separaterespective optical isomers of the derivatized amino acid mixture.
 2. Theoptical resolution method as claimed in claim 1, wherein the mixture ofoptically active amino acids is reacted with the compound at roomtemperature.
 3. The optical resolution method as claimed in claim 2,wherein the mixture of optically active amino acids is reacted with thecompound for a duration of from one to ten minutes.
 4. The opticalresolution method as claimed in claim 1, further comprising stopping thereaction between the mixture of optically active amino acids and thecompound by adding an aqueous acid solution.
 5. The optical resolutionmethod as claimed in claim 4, wherein the aqueous acid solution is anaqueous trifluoroacetic acid solution.